1. Field of the Invention
The invention is directed to a method for analyzing a specimen in which the specimen is supported so as to be rotatable around an axis of rotation and so as to be displaceable.
2. Description of Related Art
The specimen is illuminated by a first illumination device by a substantially planar light sheet substantially parallel to the axis of rotation, light radiated from the specimen is imaged as a sectional image on a detection device by an imaging objective with an optical axis which intersects the plane of the light sheet at an angle different from zero, preferably perpendicularly. In so doing, a plurality of sectional images of the specimen are recorded and the specimen is rotated and/or displaced in space between the recordings at least for a portion of the sectional images. The recorded sectional images are then registered, i.e., transferred to a common coordinate system by transformation. They are then fused to form a first data set of spatial image data of the specimen.
Further, the invention is directed to an optical arrangement for analyzing a specimen. An arrangement of this kind includes a specimen holder for receiving the specimen, the specimen and/or specimen holder being supported so as to be rotatable around an axis of rotation and so as to be displaceable. It further comprises a first illumination device which in turn comprises a first illumination light source and a illumination beam path for illuminating the specimen with a light sheet. The arrangement further comprises a detection device for detecting light that is radiated from the specimen and imaging optics which image the specimen at least partially on the detection device by means of an imaging objective in an imaging beam path, wherein light which is detected due to the illumination of the specimen by the first illumination device is detected as a sectional image, and wherein the light sheet is substantially planar in the focus of the imaging objective, and wherein the imaging objective has an optical axis which intersects the plane of the light sheet at an angle different from zero, preferably perpendicularly. Finally, the arrangement also comprises a control unit and an evaluating unit, the control unit being designed so as to control in such a way that a plurality of sectional images of the specimen can be recorded, and the specimen can be rotated and/or displaced between the recordings, and wherein the evaluating unit is designed to register and fuse the recorded sectional images to form a first data set of spatial image data.
A method and an optical arrangement of the kind described above can be applied for observation of the specimen particularly in connection with single plane illumination microscopy (SPIM), also known as selective plane illumination microscopy. Whereas in confocal laser scanning microscopy the specimen is scanned point by point in a plurality of planes at different depths and three-dimensional image information about the specimen is obtained from this, the SPIM technique is based on widefield microscopy and makes it possible to generate three-dimensional images of specimens based on optical sections through different planes of the specimen.
The advantages of SPIM include faster acquisition of images, reduced bleaching out of biological specimens, and an expanded depth of penetration of the focus in the specimen.
Basically, in the SPIM technique fluorophores which are contained in the specimen or introduced into the specimen are excited by laser light which is shaped as a light sheet or which is guided over the specimen in such a way that the shape of a light sheet results in effect, i.e., over the period of observation. Each light sheet illuminates a plane in the depth of the specimen, and an image of the specimen in this plane is obtained by means of this illumination. It is important that elements in the light sheet plane are projected on the plane of the detector, which is part of the detection device, or that the light sheet plane and detector plane are conjugate to one another. In conventional microscope constructions in which the detector plane extends perpendicular to the optical axis of the detection beam path, the direction in which light is detected is perpendicular, or at least virtually perpendicular, to the plane of illumination.
SPIM technology is described, for example, in Stelzer et al., Optics Letter 31, 1477 (2006), Stelzer et al., Science 305, 1007 (2004), DE 102 57 423 A1, and WO 2004/0530558 A1.
Another method by which spatial images of a specimen can be obtained is optical projection tomography (OPT). This method can also be used to analyze thicker specimens than is possible with the SPIM technique, but without the need to dispense with the usual staining of the specimen with dyes. A tomography device substantially comprises an illumination and imaging system—for example, an optical microscope—a detection device—generally a CCD camera—and an evaluating unit in which the recorded images are combined to form a spatial image. As in other tomography methods, the specimen must be rotated and a large number of images must be recorded at different angles, the total image then being generated therefrom by means of a reconstruction method. The more images that are recorded, the higher the accuracy. Therefore, the specimen is only rotated by a small amount, for example, by an angle of 1°, between two images. The specimen is illuminated by transmitted light, for example, with UV light or white light. Point light sources with a corresponding aperture angle can be used so that a light cone is projected onto the object. Planar light sources, i.e., light sources radiating light over a large surface area, can also be used when the light rays of the source impinge on the specimen in parallel. This corresponds to a point light source arranged at infinity. The specimen is then projected on the detector approximately corresponding to an orthogonal parallel projection. The optical axis of the illumination beam path extends perpendicular to the axis of rotation of the specimen when impinging on the specimen. Light which is emitted by a section extending perpendicular to the axis of rotation of the specimen is imaged on exactly one line of the CCD detector by the imaging optics. However, since the entire specimen is illuminated, the entire specimen is also projected on the detector in contrast to confocal microscopy. On its way through the specimen, the light is attenuated by absorption or scattering so that the projected image of the specimen corresponds to a shadow image similar to conventional x-rays, for example. The focus region of the imaging optics has the shape of a double cone whose tips touch. As a rule, the focus region does not lie in the center of the specimen, but rather along the optical axis in its front half because the focussed double cone is not symmetrical; rather, only one third of the depth of focus lies in the front region and two thirds in the rear region. The specimen can be imaged more sharply as a whole in this manner by displacing the focal point.
As opposed to other established microscopy methods and also as opposed to the SPIM technique, OPT can also acquire specimens with a thickness of up to approximately 15 mm. Observation is possible in bright field as well as dark field, which also makes it possible to stain the specimen with dyes other than fluorophores. However, the resolution is lower than in confocal microscopy.
The method of optical projection tomography is described, for example, in J. Sharpe, et al., Science 296, 541 (2002), WO2004/020996, and W02004/020997A1.
In contrast to OPT technology, however, SPIM technology is more prone to effects brought about by the scattering and absorption of light. These artifacts manifest themselves in SPIM images, for example, as stripes.